Keratan sulfate is a glycosaminoglycan and is present as a side chain of proteoglycan or keratan sulfate proteoglycan such as aggrecan, keratocan, or lumican, in limited tissues such as cartilage and the cornea. Keratan sulfate is an acidic polysaccharide having, as a basic sugar chain structure, a disaccharide recurring structure formed of N-acetyl-D-glucosamine and D-galactose, and sulfated with various degrees. In the cornea, keratan sulfate is an important component for maintaining transparency thereof, and in cartilage, keratan sulfate is an aggrecan component serving as an important extracellular matrix for maintaining the structure of cartilage. Particularly, since serum keratan sulfate levels of patients of joint diseases such as osteoarthritis and rheumatoid arthritis are known to vary, keratan sulfate is a candidate substance as a marker for diagnosing joint diseases.
Keratan sulfate is categorized into keratan sulfate-I, originating from the cornea and mackerel skin, and keratan sulfate-II, originating from cartilage, intervertebral disks, and pulpous nuclei. The mode of bonding of keratan sulfate to the core protein varies between keratan sulfate-I and keratan sulfate-II. In keratan sulfate-I, an aspartic acid residue is bonded to a sugar chain via N-glycoside bonding, while in keratan sulfate-II, a serine or threonine residue is bonded to N-acetylgalactosamine via O-glycoside bonding (Seikagaku Jiten (3rd edition), published by Tokyo Kagaku Dojin). Regarding the structural features of keratan sulfate, keratan sulfate-I has a main structure including four saccharide moieties and three sulfate groups, and keratan sulfate-II has a main structure including four saccharide moieties and four sulfate groups. That is, keratan sulfate-I has a sulfate content lower than that of keratan sulfate-II.
One known antibody against such keratan sulfate is 5D4 (name of clone). The antibody is known to recognize (or bind), as a minimum recognition unit, a structure including five sulfate groups with respect to six saccharide molecules (Non-Patent Document 1) and to react with a keratan sulfate having a relatively high sulfate content. This antibody is commercially available from Seikagaku Corporation and is widely employed in research.
Examples of currently employed keratan sulfate detection methods include cellulose acetate membrane electrophoresis, high-performance liquid chromatography (HPLC), and immunological assay (e.g., ELISA). Due to poor sensitivity, cellulose acetate membrane electrophoresis is disadvantageous for the detection of a micro-amount of keratan sulfate in a sample. One known HPLC method is a method by Miyauchi et al. (Patent Document 1). In such an HPLC method, keratan sulfate contained in the sample is digested by keratanase, which specifically decomposes keratan sulfate, and the formed disaccharide is analyzed. Although the HPLC method exhibits high specificity and higher sensitivity, the sample must be subjected to preliminary treatments such as digestion by protease, crude purification, and digestion by keratanase, generally making this method disadvantageous for the treatment of a large number of samples. The ELISA method is suitable for the treatment of a large number of samples and is highly operable. Examples of known ELISA techniques include a competitive ELISA method employing 5D4 (Patent Document 2), and the sandwich ELISA method employing 5D4 (Patent Document 3).
The competitive ELISA method, which employs only one type of antibody, exhibits low specificity in the assay system in some cases, and is thought to be affected by a substance co-existing in the sample. Thus, the sandwich ELISA method exhibits higher specificity. The keratan sulfate assay method through sandwich ELISA employing 5D4 is useful for the detection of keratan sulfate in a sample. Actual measurement results obtained through the assay have been reported for a serum or synovial fluid of humans, dogs, horses, and rabbits, and a synovial fluid of guinea pigs. However, there have been virtually no reports on the assay of rat-derived and mouse-derived samples, which are thought to contain a very small amount of keratan sulfate. Thus, even at present, keratan sulfate assay encounters difficulty, and there are many biological samples in which the presence of keratan sulfate is undetermined.